Project Summary/Abstract Our long-term goal is to understand the fundamental principles of transcriptional regulation in normal development, stress responses, and human disease. Over the past several years, my lab has focused on an investigation of the mechanisms by which cell type-specific enhancers influence gene expression programs. Using high throughput sequencing, we recently uncovered a new class of enhancers that support the production of a vast number of noncoding enhancer RNAs (eRNAs) that are expressed in a manner that correlates with changes in gene expression in response to proinflammatory signaling. We also identified several transcription factors and epigenetic modifiers that are recruited to these enhancers to control eRNA synthesis through mechanisms that we aim to elucidate in our proposed research plan. Notably, my lab also found that several of the identified eRNAs exhibit direct roles in the regulation of gene expression, and this finding is among the few number of studies that have demonstrated a function for eRNAs. These important findings revealed to us the need of implementing new technical approaches to take the next step of investigating the mechanisms by which eRNAs function since current methodologies are unable to uncouple the functions of eRNAs from the act of enhancer transcription. To address this need and to advance our understanding of the precise functions of eRNAs, we have started implementing our powerful cell free assays with cloned enhancers and genes, factors purified from cellular extracts, and synthetically transcribed eRNAs. Using this system, we have very recently uncovered an exciting mechanism by which eRNAs enhance transcriptional activation by decreasing the dissociation kinetics and thereby increasing the residence time of a key epigenetic regulator at enhancers. In the next 5 years, we aim, through the proposed research plan to unleash new frontiers of gene regulation by uncovering mechanistic principles that govern eRNA synthesis and their subsequent mechanisms of action. We will focus specifically on the identification of (i) mechanisms by which enhancer- specific epigenetic writers and histone marks regulate eRNA production. Progress toward this goal will provide new insights into histone marks that have denoted enhancers for almost a decade but their functions remain unknown and (ii) new eRNA binding partners and their functions in enhancer-dependent transcriptional regulation. Importantly, our inducible cellular system together with our cell-free assays will be employed as a paradigm to understand the events controlling eRNA synthesis and function. Importantly, the results stemming from the proposed work will readily advance our analysis of eRNAs in eukaryotic transcription in multiple new directions. This is an issue of great significance in light of the emerging field of functional eRNAs and the potential of using eRNAs as diagnostic markers and therapeutic targets for altering enhancer activities that are linked to human disease.